Project Summary Autism is a pervasive developmental disorder caused by heterogeneous insults at the cellular or molecular level affecting the function of distributed brain regions. Co-morbid anxiety and other psychiatric disorders are common, likely due to underlying mechanisms shared with core features of autism. Loss of the postsynaptic density protein Shank3 is associated with deficits in synapses at the molecular level, as well as autism and intellectual disability. Autism is associated with changes in prefrontal circuit function which likely impede the spatial and temporal patterning of neuronal ensemble activity, degrading the precision with which the prefrontal cortex responds to input. I propose to test the hypothesis that abnormal recruitment of prefrontal activity in response to vHPC input during anxiety contributes to abnormal anxiety in the Shank3 knockout (KO) mouse. I will first define the specificity with which ensemble activation occurs in response to vHPC input within prefrontal microcircuits, and determine the degree to which ensemble recruitment is altered in mice lacking Shank3. These mice are abnormally anxious and have abnormal social interaction. I have demonstrated that network organization in prefrontal slices from KO mice is abnormal; individual neurons are more active, and the organization of ensemble activity also abnormal. Specifically, pairwise correlations are abnormally high and the KO generates abnormal patterns of activity. I quantified the patterns of activity sampled by the network by counting the number of specific n-neuron motifs consisting of combinations of 2,3,4, or 5 neurons. This revealed that the KO samples a greater number of patterns, but patterns sampled are less likely to occur more frequently than in shuffled data. This suggests that while the KO samples more diverse modes of activity, it does so in a disorganized manner. This may increase noise or decrease the precision of ensemble recruitment during behavior. I propose to test how these changes in network activity affect how the PFC responds to anxiogenic input using optogenetic stimulation of defined inputs from the ventral hippocampus to examine neuronal ensembles recruited in response to specified input (Aim 1). I will then use implanted microendoscopes to explore the precision with which distinct ensembles of neurons are recruited during anxiety-related behavior (Aim 2) in the intact animal, and the precision with which anxiogenic input from the ventral hippocampus results in activation of PFC projections to the amygdala (Aim 3). These studies are of immediate relevance to autism, as despite a dramatic increase in our knowledge of genetic and cellular pathology underlying autism there remains a paucity of therapeutic options. This mentored award will provide the opportunity to develop technical skills and quantitative methods needed for the analysis of large, dynamic populations of neurons. I will be mentored by Dr. Vikaas Sohal, a clinician-scientist and an expert in optogenetics, neuropsychiatric and developmental disorders. I intend to submit an R01 and transition to the role of independent investigator by the end of this award.